1. Field
The present invention relates to a lateral flow quantitative assay system capable of quantifying glycohemoglobin in blood with high sensitivity and a method thereof, in particular, an integrated quantitative assay method and system capable of simultaneously quantifying glycohemoglobin and total hemoglobin.
2. Description of the Related Art
Diabetes mellitus is a disease of abnormal carbohydrate metabolism in which glucose cannot enter the body's cells and be utilized by the body, and therefore remains in the blood in high concentrations to cause many complications. There are three types of diabetes mellitus: The major form of diabetes is type 1 diabetes, called insulin-dependent diabetes mellitus, which occurs when the beta cells of the pancreas are damaged by an autoimmune reaction and stop producing or secreting the hormone insulin. Type 2 diabetes, called non-insulin-dependent diabetes mellitus, is characterized by peripheral insulin resistance and impaired insulin secretion. The third type is gestational diabetes which occurs typically during pregnancy. It has been known that type 2 diabetes is the most prevalent, consisting of 90 to 95 percent of diabetes patients in advanced countries, while type 1 diabetes and gestational diabetes are less common.
Diabetes can be diagnosed by measuring urine or blood glucose levels, but urine glucose is not reliable for diagnosis. Blood glucose test is also inaccurate, since blood glucose levels can be affected by several factors, including diet and exercise. For these reasons, a glycohemoglobin test is a blood test used primarily to diagnose or monitor diabetes.
In 1986, the American Diabetes Association recommended glycohemoglobin testing twice a year to monitor all types of diabetes, and thus glycohemoglobin has gained acceptance as a stable index of blood glucose control, and has been commonly used to manage diabetes, since the DCCT (Diabetes. Control and Complications Trial) showed a direct relationship between glycohemoglobin level and diabetes complications in 1993.
Based on the results of the DCCT and UKPDS (United Kingdom Prospective Diabetes Study), current ADA (American Diabetes Association) guidelines recommend a goal glycohemoglobin of less than 7%. If the levels are greater than 8%, the ADA recommends that diabetes treatment be examined and modified as needed. In 2001, the American Association of Clinical Endocrinologists recommended a target glycohemoglobin of 6.5% or less, based on the UKPDS data that the incidence of diabetic retinopathy was shown to increase at the value of 6.5% or more. In 1999, the International Diabetes Federation (IDF) also recommended a target value of 6.5%.
In accordance with the clinical studies involving 1441 patients, conducted by the DCCT for 6.5 years, the risk of microvascular complications can be markedly reduced by achieving tight blood glucose control. Therefore, it is essential that patients or physicians consider strict management of glucose levels.
Human adult hemoglobin typically consists of three types of Hb; 97% HbA, 2.5% HbA2 and 0.5% HbF. Among them, HbA consists of four polypeptide chains—two alpha chains, each containing 141 amino acids, and two beta chains, each containing 146 amino acids. Chromatographic analysis of HbA identifies 96% major Hb and 5˜6% minor Hb, termed HbA1 or glycohemoglobin. The glycated form, formed by attachment of glucose to the N-terminal valine of the beta chain, accounts for 80% of HbA1, and HbA1a and HbA1b accounts for the remaining portion.
Glycation is the non-enzymatic addition of a sugar molecule to amino groups of proteins, and is a very slow and irreversible reaction. Glycohemoglobin is formed continuously by the addition of blood glucose to hemoglobin, and the proportion of glycohemoglobin to hemoglobin is dependent upon the exposure time of erythrocyte to glucose. Specifically, during glycation, glucose is attached to the valine residue of HbA to form a HbA1c precursor, which subsequently undergoes Amadori rearrangement to form a stable ketoamide form. At this time, the increase in blood glucose levels increases the exposure time of hemoglobin to circulating glucose, resulting in the high proportion of glycohemoglobin. Thus, the percentage of glycohemoglobin reflects the blood glucose levels. In addition, the glycohemoglobin test can help monitor the long-term control of blood glucose levels, since the life span of erythrocytes ranges from 60 to 120 days.
Various methods for the determination of glycohemoglobin in blood have been developed. Currently available methods include ion exchange chromatography, affinity chromatography, electrophoresis, combined colorimetry or the like. These methods are difficult to practice, and thus require advanced skills and equipment. In addition, concerning the development trends of disposable clinical analysis systems, there have been proposed very useful quantitative assay systems for use at a remote site, at home, or point-of-care testing, such as visual, optical, and electrochemical detection methods.
Recently, monoclonal and polyclonal antibodies that recognize the N-terminal peptide residues of glycohemoglobin have been developed (U.S. Pat. No. 4,647,654), and thus many studies have been advanced on quantitative immunoassay systems for glycohemoglobin using the antibodies. Since antibodies recognizing glycohemoglobin are used in the immunoassay systems, they are advantageous in terms of specificity and sensitivity. When glycohemoglobin levels are determined by immunoassay, it is essential to prepare antibodies capable of recognizing a specific glycosylated region of glycohemoglobin with high sensitivity. Since the glycosylated region of glycohemoglobin in blood is not externally exposed, glycohemoglobin has to be first modified in order to be recognized by the antibodies. Subsequently, hemoglobin has to be converted into methemoglobin for spectroscopic measurement of total hemoglobin. Methemoglobin has a property of absorbing light at a specific wavelength, and thus its absorbance is measured by a spectroscopic method to quantify total hemoglobin concentration. In addition, modified glycohemoglobin is measured by an immunological method to quantify the amount of glycohemoglobin in blood.
Immunoassay devices are divided into flow-through type and lateral flow type according to their principle. In the flow-through type, an antibody is covalently coupled to the surface of a porous matrix, and an analyte in a sample binds with the immobilized antibody. Subsequently, a secondary capture antibody is added thereto, followed by visual detection with chromogenic enzyme substrates. There are two types of lateral flow immunoassay devices; one type is an all-in one device, and the other type is a device, in which a fluid sample binds with a labeled antibody, while passing through a labeled antibody-immobilized porous matrix.
The lateral flow type has a structure comprising a sample pad, to which a sample is applied, a releasing pad coated with a detector antibody, a developing membrane or strip, through which components of the sample move to be individually separated and to undergo antibody-antigen reaction, and an absorption pad which is provided to continuously absorb fluid so as to cause the sample to continue moving through the device. The lateral flow assay can be widely and conveniently used in various fields such as pregnancy diagnosis, cancer diagnosis, and microbe detection. However, since quantification cannot be performed with the naked eye and hence, an exact amount of an analyte cannot be determined, its application is restricted.
Immunoassay using antibodies against HbA1c generally use a principle of determining the change of turbidity of a reaction system. In this regard, since the HbA1c-specific epitope only occurs once at the N-terminus of a beta chain of the glycohemoglobin, there is no aggregation of the antigen-antibody complex. Therefore, a polyhapten having several epitopes is reacted with an antibody to form an insoluble immune complex. This can be measured turbidimetrically. The turbidimetric signal is inversely proportional to the concentration of glycohemoglobin in the sample. However, this method is disadvantageous in that a specialized analytical laboratory and automated equipment are required, since it comprises several processing steps.
Korean Patent Publication No. 2004-0018893 discloses a glycohemoglobin test kit, which consists of a buffer solution containing antibodies against glycohemoglobin, a strip containing antibodies against hemoglobin, and a washing solution. Unfortunately, an exact amount of glycohemoglobin cannot be determined, since the kit is a semiquantitative assay system, in which a dye is added to the antibodies against glycohemoglobin, and color change caused by the presence of HbA1c is detected by the naked eye or by visual comparison against a color chart.
The RIA or ELISA method which can quantify an analyte at present involves several complicated steps for such quantification, including treatment with an enzyme and washing. Therefore, there is a great demand for a general assay method which can perform quantification more rapidly, conveniently and sensitively.